The nucleotide sequences of a DNA molecule carry an information code which can be transcribed into an intermediate messenger (mRNA) followed by translation into a protein. Such a protein may have a catalytic or a structural function in the organism. In some cases the nucleotide sequence is transcribed into an RNA molecule and this RNA molecule is the functional entity. Natural examples are the ribosomal RNA molecules (rRNA), transfer RNA molecules (tRNA) and small nuclear RNA molecules (snRNA). Some viruses (e.g., retroviruses) are reverse transcribed and the DNA product is then incorporated into the host genome from whence it can be transcribed once again into viral RNA. A useful artificial situation would be to isolate a DNA fragment of exactly the same sequence as a useful RNA molecule and then to insert such a fragment precisely at the transcription initiation site downstream from a strong promoter sequence which in turn has been inserted into a vector plasmid. In such a case large quantities of the desired RNA molecule could be produced by in vitro transcription. Prior vector systems have provided insertion sites downstream from a promoter, but the RNA transcribed therefrom contains extraneous sequence at the 5' end. The function of the resulting RNA may be modified in an undesired or uncontrolled manner since many RNA functions are affected by the nucleotide sequence at the 5' end. This prior art difficulty could be overcome by a transcription vector providing for precise initiation of transcription of the inserted DNA segment. In this manner it is possible to produce wild type viral RNA sequences, mutated viral RNA sequences, viral RNA sequences in which some nonessential nucleotide sequences have been replaced by useful foreign genes and wild type or mutated rRNA, tRNA or sRNA molecules. Mutated viral RNA sequences may be used to infect plant cells thereby preventing superinfection or viral RNA sequences partly replaced by foreign genes may be used as vehicles for the introduction of those genes into plant cells. Alternatively the metabolism of microbial populations may be disrupted by transformation of the population with the recombinant plasmid carrying mutated rRNA, tRNA or snRNA.
In the past several years a very large potential has developed in the field of genetic engineering for the transfer of foreign genes to plants. In order to achieve such a transfer, it is necessary to find suitable vectors. The most advanced work on transfer of genes to a plant genome has been achieved by the use of Agrobacterium tumefaciens and, to a lesser extent, Agrobacterium rhizogenes (Leemans, J., et al. (1982) in Kahl, G. and J. S. Schell (1982) Molecular Biology of Plant Tumors Ch. 21:537-545, Academic Press, New York). A recent study (Murai, N., et al. (1983) Science 222:476-482) showed that sequences coding for the bean seed protein phaseolin were inserted into the sunflower genome by the transferred DNA regions of tumor inducing plasmids. In one instance the phaseolin encoding sequences were controlled by the octopine synthase promoter and in another instance by the phaseolin promoter region. In both cases the phaseolin genes were correctly transcribed, processed and translated, thus demonstrating the expression of a plant gene after transfer to a taxonomically distinct botanical family.
In addition, there is the potential of genetically engineering the normal plasmids of Rhizobium strains prior to the infection and formation of nodules on plant roots. Much work has already been done on the structure and function of nitrogenase genes (Scott, K. F., Rolfe, B. G. and J. Shine (1983) DNA 2:141-148; Scott, K. F., Rolfe, B. G. and J. Shine (1983) DNA 2:149-156). The promoter of the nitrogenase genes of these organisms have been used to control the transcription and translation of a variety of foreign genes inserted into the symbiotic plasmids of Rhizobium in such a manner as to be under the control of these promoter regions (EPO Publication No. 0,130,047, published Jan. 2, 1985). Both types of vectors, i.e., the T-DNA of Agrobacterium spp. and the symbiotic plasmids of Rhizobium spp., involve the transfer of foreign genes as DNA.
Another possibility for the transfer of foreign genes to plant cells is by the use of RNA or DNA viruses. For retroviruses, which replicate their RNA through DNA intermediates, naturally occurring infectious viral DNA forms can be isolated (J. O'Rear et al. (1980) Cell 20:423). Other RNA viruses replicate their genomes without passing through a DNA stage. In the case of these viruses, it would be necessary to reverse transcribe them into cDNA molecules because of the technical difficulties of handling RNA molecules and the lack of restriction endonuclease active on RNA. In some cases where amplification by secondary infection cycles is possible, e.g., poliovirus (Racaniello, V. and D. Baltimore (1981) Science 214:916-919) and potato spindle tuber virus (Cress, D., Kiefer, M. and R. A. Owens (1983) Nucleic Acids Res. 11:6821-6835), the method appears feasible, but in many examples, infectivity of viral cDNA molecules is low or nonexistent, and this approach does not produce results. Despite attempts in a number of laboratories, complete genomic cDNA clones from a range of other RNA viruses have not yet proved directly infectious. To understand why these negative results may occur, it is useful to list the following probable requirements for infectivity of directly-inoculated viral cDNA:
(1) cDNA uptake into the cell; (2) cDNA transport into the nucleus; (3) transcription of (at least) full-length viral RNA's (vRNA); (4) avoidance of vRNA splicing; (5) processing of vRNA termini to infectious forms; (6) vRNA transport to the cytoplasm; (7) translation of (at least some) viral proteins; and (8) effective interaction of vRNA with viral and cellular proteins for a first round of replication.
Although effective transcription is often considered to be the most critical feature, viral cDNA infectivity probably involves a number of additional requirements. For example, linkage of viral cDNA to a strong plant promoter might be a useful strategy but would not give infectivity if the viral RNA contains a cryptic splice site which leads to its effective processing to a non-infectious form. Also, a number of steps, e.g. (4) to (6) above, involve cellular processes which are poorly understood. Consequently, the infectivity of directly-inoculated cDNA could be severely limited at a number of steps, e.g., vRNA transport to the cytoplasm. These defects of infectivity would be extremely difficult to identify and so the various defects would be very difficult to correct without significant advances in basic molecular and cellular biology.
Several features are needed to turn a fragment of nucleic acid into a plant vector: (1) The nucleic acid should be capable of being cloned so that useful quantities can be recovered; (2) The nucleic acid should be able to replicate within a plant cell and preferably, should be recognizable by selective methods; (3) It should not be pathogenic; (4) The nucleic acid must be capable of incorporating other nucleic acid sequences into its structure and of expressing such incorporated nucleic acid; and (5) If heritable changes of the transformed plant are wanted, then the vector or a derivative of the vector should be stably maintained from one generation to the next. Further, in the case of T-DNA, at some stage the transformed plant cell will have to be purified by cloning and regenerated into a plant. When these features are considered in relation to RNA plant viruses, a number of difficulties become apparent: (1) The techniques of genetically manipulating and recombining RNA are much more difficult than the techniques of genetically manipulating and recombining DNA; (2) Since most virus particles are packaged into protein capsids, there may be difficulties in accommodating more than limited quantities of "foreign" nucleic acid; (3) RNA viruses need to be replicated within the plant cell so there will be difficulties in recovery of genetically engineered RNA; (4) The strain of RNA viruses used to infect the plant cells should cause minimal pathogenic effects.
The majority of known viruses infecting eukaryotes encapsidate RNA genomes. This is particularly true among plant viruses, where 24 of 28 recognized groups produce particles containing RNA (R. E. F. Matthews (1982) Intervirology 17:1). Cloning and manipulating cDNA copies of such viral RNAs has greatly facilitated progress in RNA virology in recent years. However, use of recombinant DNA technology in the study of most RNA viruses has been seriously limited by inability to express infection from viral cDNA clones. Methods to overcome this limitation by constructing complete viral cDNA clones from which infectious products can be produced by in vitro transcription are needed. Expression of viral cDNA by such methods allows detailed molecular genetic analysis of RNA virus replication, gene expression, and regulation and the construction of practical expression vectors based on RNA viruses.